This proposal addresses specific mechanistic questions about general biological priniciples utilizing a powerful genetic system. We wish to continue our studies on the "Structure of the Subunits of Eukaryotic Chromosomes" by focusing attention on non- nucleosomal elements that define the functional boundaries of genes and chromosomal loops. For all of these studies we have selected Saccharomyces cerevisiae as the system of choice. Our experimental approach combines biochemical, cell biological, immunological, genetic, and reverse genetic techniques to address the four following Specific Aims: (1) To determine the relationship between the cis-acting DNA sequences necessary for creating a nuclease hypersensitive region in chromatin at the 5'-end of a gene and for mediating transcriptional initiation. (2) To determine the relationship between the cis-acting DNA sequences necessary for establishing an abrupt transition in chromatin structure at the 3'-end of a gene and for mediating transcriptional termination. (3) To delineate the evolutionarily conserved elements participating in the "chromosomal loop attachment" of a mouse MAR sequence ("matrix association region") that has been introduced into yeast. o precise DNA sequences o role of topoisomerase II o other DNA binding proteins o intranuclear location of binding-sites (4) To determine the functions of homologous MAR elements in yeast.